Generation of insulin-producing pancreatic β cells from multiple human stem cell lines

Source

Six-stage directed differentiation protocol (~34 days + 1-2 weeks for stem cell expansion and final assessment)
Starting material: 10 hPSC lines (5 new + 5 previously published) on Matrigel-coated TCP
Key innovation: fully planar culture (no 3D suspension required)
Stages:
Stage 0: hPSC culture in mTeSR1
Stage 1 (4 d): Definitive endoderm — Activin A + CHIR99021
Stage 2 (2 d): Primitive gut tube
Stage 3 (2 d): Pancreatic progenitor (PDX1+)
Stage 4 (4 d): PDX1+/NKX6-1+ pancreatic progenitor — KGF + SANT1 + TPPB + LDN193189 + RA
Stage 5 (7 d): Endocrine induction — latrunculin A (24 h) + XXI + T3 + ALK5i + SANT1 + RA
Stage 6 (14+ d): SC-β cell maturation
Final step: optional aggregation into islet-like clusters on orbital shaker

Eliminates the previous requirement for 3D suspension culture to generate functional SC-β cells.
Key mechanistic insight: actin cytoskeleton state controls NEUROG3 endocrine induction.
TCP monolayer culture → high actin polymerization → prevents premature NEUROG3, enables NKX6-1 expression (good).
But polymerization also blocks subsequent endocrine specification — solved by 24 h latrunculin A depolymerization at the start of stage 5.
Planar methodology is more reproducible across multiple hPSC lines (many lines don't adapt well to suspension culture).
Simplifies differentiation significantly — removes need for spinner flasks or Transwells.
Produces 0.5-0.75 million cells/cm² with 2-3× higher cell yield per media volume vs. suspension protocols.
Transplanted cells rapidly reverse severe preexisting diabetes in mice.

First pancreatic islet entry in the collection — fills major gap.
Represents a new generation of "simplified" hPSC differentiation protocols that prioritize cross-line robustness over 3D architecture.
Should be read alongside Balboa 2022 which focuses on maturation / functional benchmarking.

Establishes baseline pancreatic islet protocol for the collection.
Complements Balboa 2022 (functional maturation benchmark) and Koike 2021 (hepato-biliary-pancreatic multi-organ model).

How does planar SC-β functional maturity compare to Balboa's optimized suspension protocol?
Can the latrunculin A insight transfer to other endocrine differentiation protocols?
What is the upper limit of batch-to-batch reproducibility in the planar approach?

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